We have developed primary astrocytes culture with either RB-TS inactivation alone (T) or combined RB-TS inactivation and PTEN deletion (TP). Consistently, our preliminary data showed that both T and TP cells could not give rise to solid tumor mass upon orthotopic injection into syngeneic mice. Human and mouse EGFR VIII or EGFR wt driving by CMV promoter was constructed and plasmid DNA was transfected into T and TP cells. Stable clones will be selected and injected orthotopically into mouse cortex to determine if tumor mass will be formed upon EGFR overexpression. We are in the process of selecting stable clones for injection. In addition we are testing the roles of PDGFalpha expression and NF1 inactivation.